Integrin-alpha-M-beta-2 (CD11b/CD18, Mac-l) plays a pivotal role in the inflammatory response and host defense. By mediating critical adhesive reactions of neutrophils it participates in regulating neutrophil influx to the sites of inflammation and initiates numerous neutrophil responses. This receptor is also a potential therapeutic target in many diseases in which the inflammatory component plays an essential role. Alpha-M-beta-2 exerts its functions by binding a multitude of biologically diverse molecules. However, the mechanism which allows alpha-Mbeta-2 to exhibit broad specificity and the biological value of alpha-M-beta-2 ligand promiscuity are not known. Our long term goal is to understand the molecular basis for ligand recognition by alpha-M-beta-2, and to determine how alpha-M-beta-2 ligand promiscuity affects its adhesive functions. Studies over the past funding period have mapped the alpha-M-beta-2-binding site in the physiologically important ligand fibrinogen, and identified the recognition peptide that effectively disrupted alpha-M-beta-2-mediated cell adhesion not only to fibrinogen but also to many other ligands. Based on these findings, we have hypothesized that the fibrinogen recognition motif contains critical structural information required for alpha-M-beta-2 binding and that it may serve as a prototype adhesive signal in many alpha-M-beta-2 ligands. We will test this hypothesis by analyzing alpha-M-beta-2-binding sites in fibrinogen and in other physiological ligands using combinatorial peptide libraries and mutational analyses of fibrinogen domains. We will also use phage display to delineate the general principles that control alpha--beta-2 ligand recognition. Our data further indicated that a discrete region in the alpha-M-I-domain of alpha-M-beta-2 participates in binding of numerous ligands which has led us to propose the hypothesis that the unique region in the alpha-M-I-domain constitutes the consensus binding site for recognition of many ligands. Through the use of chimeric and point mutations between alpha-M-beta-2 and alpha-L-beta-2, the related beta-2 integrin with a different ligand specificity, we will determine the structural features required for the interaction of the consensus binding site with multiple ligands. Finally, the impact of alpha-M-beta-2's multi-ligand binding potential on its adhesive functions will be studied. We hypothesize that the alpha-M-beta-2 ability to be upregulated to high density on the surface of neutrophils and its capacity to engage numerous ligands in the extracellular matrix may modulate general cell adhesiveness and, thus, stop neutrophil migration at the site of inflammation. We will test this hypothesis by manipulating alpha-M-beta-2 expression on the surface of neutrophils and will also use neutrophils with alpha-M-beta-2 deficiency. Together, these studies will provide important insights into the structural basis of alpha-m-beta-2 ligand binding and define the mechanism by which alpha-M-beta-2 binding promiscuity controls leukocyte adhesive functions. Thus, these studies may lead to an increased understanding of the principles that govern ligand recognition by integrins and will be useful in the design of novel therapeutic agents to specifically disrupt alpha-M-beta-2-ligand interactions